Characterization of monoclonal antibodies against Chlamydomonas flagellar dyneins by high-resolution protein blotting.

Abstract

Monoclonal antibodies that recognize individual polypeptides of the outer arm dyneins of Chlamydomonas flagella were obtained and used to study the structural relationships between the various polypeptides. Immunoblot analysis showed that the y heavy chain of 12S dynein and the a and /3 heavy chains and M, 69 The dynein heavy chains migrate very close to one another in NaDodSO4/polyacrylamide gels and are well resolved only at very low protein loadings (2). Consequently, characterization of our antibodies by immunoblot analysis required (i) a reliable procedure for efficiently transferring high molecular weight polypeptides from NaDodSO4/polyacrylamide gels to nitrocellulose sheets without loss of resolution and (i) a sensitive method for detecting the transferred proteins on the nitrocellulose. The techniques that we have developed to fulfill these requirements were essential for unambiguous identification of the chains recognized by our antibodies and should be useful to a wide range of investigators needing to electrotransfer high molecular weight proteins or to detect nanogram amounts of protein on nitrocellulose. MATERIALS AND METHODSPreparation of Axonemes and Purification of Dynein. The isolation of flagella from Chlamydomonas reinhardtii (wildtype strain 1132D-) by the dibucaine method, preparation of axonemes, extraction with 0.6 M KCl, and subsequent purification of 12S and 18S dyneins by sucrose gradient centrifugation and hydroxylapatite column chromatography were as described (2, 4).Ram sperm tails were prepared from ejaculated sperm by modifications of methods described by Tash and Means (6) and Harrison (7). A pellet containing sperm tails and midpieces was treated with 0.05% Nonidet P-40 (BDH) in 30 mM Hepes, pH 7.5/5 mM MgSO4/1 mM dithiothreitol/0.5 mM EDTA/25 mM KCl/1 mM phenylmethylsulfonyl fluoride to remove the membranes. The demembranated axonemes were then collected by centrifugation at 30,500 x g and resuspended in electrophoresis sample buffer.Axonemes and latent-activity dynein-1 (LAD-1) from T. gratilla were the gift of

Differential Light Chain Assembly Influences Outer Arm Dynein Motor Function

“…Other antibodies used in this study were blot-purified rabbit polyclonal antibodies R5932 (vs. LC1; Benashski et al, 1999), R5391 (vs. LC2;Patel-King et al, 1997), R4930 (vs. LC3;Patel-King et al, 1996), CT61 (vs. LC4; M. Sakato and King, unpublished data), R4929 (vs. LC5;Patel-King et al, 1996), R4928 (vs. LC6; King and Patel-King, 1995), R7178 (vs. LC7a;Bowman et al, 1999), CT116 (vs. LC7b;DiBella et al, 2004a), R4058 (vs. LC8; King and Patel-King, 1995), and anti-DC 2 (Wakabayashi et al, 2001), and mouse monoclonal antibodies 12␥B (vs. ␥ HC; King et al, 1985), 1878A (vs. IC1; King et al, 1986), and 1869A (vs. IC2; King et al, 1985). Antibody reactivity was detected using peroxidase-conjugated secondary antibodies and an enhanced chemiluminescent substrate (GE Healthcare, Little Chalfont, Buckinghamshire, United Kingdom).…”

“…Previously, we observed that both IC1 and IC2 yielded products of M r 82,000 and 91,000, respectively, after EDC treatment (King et al, 1991;DiBella et al, 2004a), and the M r of these LC9-containing products suggested that they may derive from attachment of LC9 to both IC1 (76,623 Da) and IC2 (63,519 Da). To test whether these bands indeed represent LC9-IC complexes, blots of dynein samples treated with 20 mM EDC were cut lengthwise and one-half probed with CT231 and the other with either 1878A (vs. IC1; King et al, 1986) or 1869A (vs. IC2; King et al, 1985) (Figure 4, middle and right). After reassembly of the blots, we found that the lower and upper LC9-containing bands precisely comigrated with bands detected by 1869A and 1878A, respectively.…”

Oda5p, a Novel Axonemal Protein Required for Assembly of the Outer Dynein Arm and an Associated Adenylate Kinase

“…Oda5p was localized using the anti-Oda5p antibody diluted 1:1000 in 5% horse serum in 1ϫ Tris-buffered saline/0.5% Tween 20. IC2, the ␥DHC, DC2, and IC140 were revealed using monoclonal antibodies 1869A and 12␥B (King et al, 1985), an anti-DC62 polyclonal antibody (Wakabayashi et al, 2001), and an anti-IC140 polyclonal antibody (Yang and Sale, 1998), respectively. Horseradish peroxidase-conjugated secondary antibodies (Pierce Chemical, Rockford, IL; Sigma-Aldrich) were used at 1:2000.…”

The Monoclonal Antibody 1F6 Identifies a pH-dependent Conformational Change in the Hydrophilic NH2 Terminus of NhaA Na+/H+ Antiporter ofEscherichia coli

“…24. For Western blot analysis (25) proteins were transferred onto a nitrocellulose membrane using a semi-dry blotting apparatus by applying 50 mA/gel for 1 h at 4°C. After transfer, the membrane was blocked overnight in phosphate-buffered saline buffer containing 3% (w/v) BSA, 0.1% (w/v) Triton X-100.…”